Spacer arms are essential in many areas of modern biochemistry. Spacer arms can be defined as molecules which link one molecule to another molecule or to an inert support. Polyethylene glycol, for example, has been used advantageously to link enzymes to insoluble carriers and other biomolecules while retaining the activity of the enzyme. M. Stark and K. Holmberg, Biotech. and Bioeng., 34:942-950 (1989). This concept has important consequences for industrial processes using immobilized enzymes (e.g., affinity column purification processes), and for diagnostic assays (e.g., ELISA assays). Two other areas in which polyethylene glycol spacer arms have been used are peptide synthesis and sequencing. The coupling rate of protected nucleotide and amino acid residues to inert supports, such as silica, membrane, and polystyrene supports, often increases commensurately with the separation of the reaction site from the support backbone. Similar effects have been shown for sequencing of solid-phase immobilized samples. J. K. Inman et al., In Solid Phase Methods in Protein Sequence Analysis, Previero and Coletti-Previero, (Eds.), Elsevier, North-Holland Biomed. Press, pp. 81-94 (1977).
The effectiveness of solid-phase nucleic acid or peptide synthesis or sequence analysis is affected by the solid-phase or support which anchors the reactive sites. Polystyrene gels or porous glass have both been utilized as solid supports for peptide sequencing, for example. In many applications, the solvents used in the process can cause the polystyrene particles to change in volume, which causes blocking of the reaction column and back pressure. Conversely, porous glass is completely rigid and does not change in volume, but the chemical properties of porous glass derivatives have lacked reproducibility. Polymer particles, such as polystyrene particles, which have been derivatized so that reactive groups can be attached to them, have proved useful in many applications. Polyethylene glycol (PEG) structures have been used as chemically inert spacer arms because they are compatible with a wide range of solvents. Inman et al., ibid. The use of PEG spacer arms minimizes the steric effects caused by the support. PEG spacer arms provide another useful function in modifying the character of the pore space so that the support-bound reactive moiety is compatible with a wider range of solvents and reagents.
PEG-modified polystyrene (PEG-PS) resins have been described for use in solid-phase peptide sequencing. Inman et al., ibid. PEG-PS resins have also been utilized as phase transfer catalysts. W. M. McKenzie et al., J. Chem. Soc. Chem. Commun., p. 541-543 (1978); S. L. Regen et al., J. Amer. Chem. Soc., 101:116-120 (1979); J. G. Heffernan et al., J. Chem. Soc. Perkin II, p. 514-517 (1981); Y. Kimura et al., J. Org. Chem., 48:385-386 (1983); M. Tomoi et al., Reactive Polymers, 10:27-36 (1989). PEG-PS resins have been described as supports for solid-phase peptide synthesis. Becker et al., Makromol. Chem. Rapid Commun., 3:217-223 (1982); H. Hellermann, et al., Makromol. Chem., 184:2603-2617 (1983). However, PEG-PS resins prepared by the referenced methods suffer from several drawbacks. The reactions proceeded poorly with high molecular weight PEG (e.g., greater than 400 daltons) and symmetrical bifunctional PEG tended to form crosslinks. These problems were reduced by the anionic polymerization of ethylene oxide directly onto crosslinked polystyrene. Bayer et al., In Peptides: Structure and Function, V. J. Hruby and D. H. Rich (eds.), Proc. 8th Am. Peptide Symp. pp. 87-90, Pierce Chem. Co., Rockford, Ill. (1983). Bayer and Rapp, German Patent DE 3500180 Al (1986). However, the PEG chain lengths are difficult to control using this method, and the uniformity of the PEG polymers is uncertain. Another problem with this process is that the polystyrene is functionalized using chloromethyl ether, which is highly toxic, and residual chloromethyl groups can cause side reactions during peptide synthesis.
Another method of making PEG graft copolymers is described by Zalipsky et al., In Peptides: Structure and Function, C. M. Deber, V. J. Hruby and K. D. Kopple (eds.), Proc. 9th Am. Pep. Symp., pp. 257-260, Pierce Chem. Co., Rockford, Ill. (1985). In this method, certain heterobifunctional PEG derivatives of defined molecular weight (i.e., 2000 to 4000 daltons) were used. However, these derivatives are not readily available, which hinders their commercialization.
A method of preparing non-toxic and efficient solid supports which can be used with a wide range of solvents would be valuable for use in solid-phase synthesis or sequencing of peptides or nucleic acids, or for other solid-phase applications.